Polynucleotide hybridization assays using a polynucleotide probe for verifying the presence of a target polynucleotide analyte are well known in the art. Hybridization is based on complementary base-pairing between target and probe sequences. The quantity of the polynucleotide probe, as compared to the target polynucleotide, is in excess with ratios of 6:1 of probe per target, or greater being employed conventionally. Furthermore, the amount of the probe which hybridizes to the target is typically a fraction, 0.1%. Therefore, it is very desirable to quench the fluorescence of the unhybridized probe so that minute quantities of the polynucleotide probe hybridized to the target can be accurately detected.
In U.S. application Ser. No. 06/808,757, now abandoned, a homogeneous assay method is disclosed in which a polynucleotide probe is used to detect the presence of a target polynucleotide in a sample. Much of the contents of U.S. application Ser. No. 06/808,757, now abandoned has already been published. See, e.g., corresponding European Patent Application Publication No. 231,495, published on Aug. 12, 1987.
The disclosed probe comprises a polynucleotide and at least a first entity and a second entity. The first entity is attached to a first nucleotide of the polynucleotide by means of a linker arm, separated by about ten nucleotides from the second entity which is attached to a second nucleotide, also by means of a linker arm. Upon hybridization of the probe to the target polynucleotide, the entities, by virtue of their characteristics, e.g., the ability to intercalate into a target-probe hybrid, provide for a property change in either the probe, the target, or both. The change in property can be detected by appropriately measuring an associated signal, e.g., radiation emission, interaction of molecular dispersion forces, such as melting temperature, and buoyant density.
It has now been discovered that the signal intensity in a hybridization assay can be significantly enhanced by contacting the hybridization reaction mixture with a background- reducing reagent which chemically modifies the intercalating groups, e.g., of an aromatic dye, when the probe to which these intercalating groups are attached remains unhybridized to target. By modifying the intercalating groups in this way, interfering signal from the unhybridized single-stranded probe can be reduced significantly, if not altogether eliminated.